YAKUGAKU ZASSHI 127(5) 889―896 (2007)  2007 The Pharmaceutical Society of Japan 889

―Regular Articles― The Extract of pentaphyllum Enhanced the Production of Antibodies and Cytokines in Mice

Wen-Chung HUANG,a,b Ming-Ling KUO,c Ming-Liang LI,a Rong-Chi YANG,d Chian-Jiun LIOU,b and Jiann-Jong SHEN,e

aThe Department of Life Science, National Taiwan Normal University, 88 Sec. 4, Ting-Chou Road, Taipei 116, Taiwan, bChang Gung Institute of Technology, 261 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan, cDepartment of Microbiology and Immunology, Graduate Institute of Basic Medical Science, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan, dDepartment of Chinese Herbal Pharmacy, Chang Gung Memorial Hospital, 123 Dung-Hou Road, Kwei-Shan, Tao-Yuan 333, Taiwan and eSchool of Traditional Chinese Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan

(Received January 11, 2007; Accepted February 19, 2007)

Gynostemma pentaphyllum is a popular herbal tea in China and some Asian countries. The modulatory function of G. pentaphyllum total extracts on immune cells was evaluated in this study. The extract was intraperitoneally in- jected into mice for 5 consecutive days. The production of antibodies from B cells or cytokines from T cells was deter- mined mainly with ELISA. After the treatment, serum IgM and IgG2a were signiˆcantly enhanced and showed dose- dependent eŠect. Moreover, serum IgA and IgG1 were also increased when received the extract at the doses of 0.05 or 0.50 g/kg/day. In addition to the serum levels, the injection of the extract enhanced the production of all antibodies from LPS-activated spleen cells. Furthermore, more cytokines were secreted from Con A-stimulated splenocytes of G. pentaphyllum-treated mice. Our results suggest that the extract of G. pentaphyllum might promote immune responses through the activation of T and B cells.

Key words―Gynostemma pentaphyllum; antibodies; cytokines; mice

hanced plasma insulin levels in rats with hyperglyce- INTRODUCTION mia. G. pentaphyllum wasreportedtoactivateim- Gynostemma pentaphyllum () is one mune response for cancer patients.8) of the widely used herbal medicines in Southeast Chi- The function of T and B lymphocytes are most im- na. It is a perennial liana which grows in wild ˆelds in portant for host adaptive immunity. The antibodies Southern China, Japan, and Korea. In China, it is that are secreted by B cells block infectious microbes named as ``Jiao-Gu-Lan'' and usually used as an her- or foreign antigens. Among the subtypes of antibo- bal tea. According to the principles of traditional dies, IgM appears ˆrst in primary response when the Chinese medicine, the use and expecting clinical hosts encounter antigens.9) Consequently, the eŠects of G. pentaphyllum is very similar to Panax production of speciˆc IgG is able to neutralize the mi- . Indeed, about 90 kinds of gypenosides have crobes or antigens. Nowadays, it is well accepted that been also isolated from G. pentaphyllum.1) In addi- the cytokines produced form CD4＋ T cells instruct tion, the structures of more than six gypenosides from the patterns of antibody subtypes.10) Activated CD4＋ G. pentaphyllum were reported to be the same with helper T (Th) cells are mainly divided into two sub- the ginsenosides from P. ginseng.2) populations, including Th1 and Th2 cells,11) based on In recent years, several pharmacological eŠects of the cytokine expression patterns. Th1 cells secret G. pentaphyllum have been reported, such as anti- more interleukin (IL)-2,IL-12,andinterferon(IFN) cancer,3) anti-gastric ulcer,4) treatment of hepatitis,5) -g;12) however, Th2 cells produce more IL-4, IL-5, and hyperlipidaemia.6) Moreover, insulin releasing and IL-13.13) IL-10 had been grouped as Th2-type substance had also been isolated from this plant.7) cytokine, but it is now considered as the regulatory This substrate improved glucose tolerance and en- cytokine.14) Although G. pentaphyllum have been used widely e-mail: ef2013＠adm.cgmh.org.tw in several countries and some clinical eŠects have 890 Vol. 127 (2007) been approved, its modulatory function on T and B immunoglobulin concentrations in the culture super- cells has not been evaluated. With the introperitoneal natants were determined using the ELISA technique. injection of the G. pentaphyllum extract into mice, To determine the eŠects of G. pentaphyllum on the the aim of this study was to examine the eŠect of the cytokine production, the spleen cells (1×106 cells/ extract on the serum immunoglobulin levels or the ml) were cultured in the presence of 1 mg/ml con- cytokine production from active spleen cells. canavalin A (Con A) for two days. Culture super- natants of Con A-activated spleen cells were collect- MATERIALS AND METHODS ed, and the cytokine concentrations were measured Preparation of G. pentaphyllum Extracts Gy- using the cytokine-speciˆc ELISA technique. nostemma pentaphyllum total plant extracts were pre- ELISA Assay To determine the concentrations pared by the Department of Chinese Herbal Pharma- of IgA, IgG, IgM, IgG1, and IgG2a, we used sand- cy, Chang Gung Memorial Hospital. Brie‰y, dried wich enzyme-linked immunosorbent assay (ELISA) herb was soaked in water and boiled for 50 minutes. technique. Microtiter plates were coated with 100 ml The decoction was then lyophilized and was reconsti- of rabbit anti-mouse IgG＋IgA＋IgM, anti-IgG1, or tuted with phosphate buŠered saline (PBS) before anti-IgG2a antibodies (Zymed Laboratories, San use. The solution was shaken overnight and steriled Francisco, CA, USA) in PBS. After an overnight in- with 0.25 mm ˆlters, aliquoted and stored at a －80°C cubation at 4°C, the plates were washed and blocked freezer. with 1％ gelatin for 1 h at 37°C. After washing, 100 ml LPS Assay Owing to hope to know whether G. of serial dilutions of standard IgG, IgG1, IgG2a, IgA, pentaphyllum was contaminated by sold gram-nega- IgM and dilutions of serum were added, then incu- tive bacterium. We used Limulus Amebocyte Lysate- bated for 2 h at 37°C. Consequently, a horseradish had kit to assay LPS content (Cape Cod, E. peroxidase (HRP)-conjugated secondary antibody Falmouth, MA, USA). LPS was deˆned that was (Zymed Laboratories) was added followed with the contaminated over 0.5 EU/ml in the test.15,16) incubation of 100 mlofo-phenylenediamine (OPD) Animals and the Administration of G. pentaphyl- solution (0.4 mg/ml) for 20 min at room tempera- lum Extracts Male BALB/cmiceaged68 weeks ture. Finally, the reaction was stopped by 25 mlof3N

(average weight: 2025 g) were purchased from H2SO4. The optical density at 490 nm in each well was Laboratory Animal Center, College of Medicine, Na- read using an ELISA reader. tional Taiwan University. Mice were accommodated The amounts of interleukin (IL)-2, interferon one week before the experiments and maintained and (IFN)-g,IL-4andIL-10weremeasuredwiththeuse handled according to the guidelines of Animal Care of ELISA kits speciˆc to each cytokine (R&DSys- Committee of Chang Gung University and NIH tems, Minneapolis, MN, USA). The minimum de- Guides for the Care and Use of Laboratory Animals. tectable concentration is 9.4 pg/ml for IFN-g,15.6pg Mice were divided into four groups (six mice per /ml for IL-2, 7.8 pg/ml for IL-4, and 15.6 pg/ml for each group) and injected intraperitoneally with nor- IL-10. mal saline (as normal control) or various doses of G. Statistics Analysis Thedatawereanalyzedus- pentaphyllum extracts, including 0.05 g, 0.5 g, and ing One-way analysis of variance (ANOVA) with 5.0 g per body weight daily (g/kg/day) for 5 con- post-hoc Dunnett's test. Values were presented as the secutive days. The experiment was repeated twice. mean±the standard error (SE). Probability values Serum Collection and Splenocyte Cell Cultures (p) of less than 0.05 were considered to be sig- Mice were sacriˆced on day sixth. The blood was har- niˆcant. vested and centrifuged at 6,000 rpm for 20 min. The RESULTS serum was then collected and stocked at －80°C. Single cell suspensions were prepared as previously G. pentaphyllum Induced Higher Levels of Im- described.17) The spleen cells (5×105 cells/ml) were munoglobulin in Mice Serum Each group of cultured in a medium containing RPMI 1640 sup- BALB/c mice received intraperitoneal injection of plemented with 10％ fetal bovine serum (FBS),2mM various doses (0, 0.05, 0.5, or 5 g/kg/day) of G. pen- L-glutamine, 100 U/ml penicillin/streptomycin, and taphyllum for 5 consecutive days and then were 1 mg/ml lipopolysaccharide (LPS) for ˆve days. The sacriˆced on day 6. The mice were designated as Nor- No. 5 891 mal, 0.05 G, 0.50 G, and 5.00 G groups, respectively. tively). The highest dose of G. pentaphyllum (5.00 LPS assay of high dose 5.00 G group was about 0.192 G), however, suppressed the serum IgG1 level (0.65 EU/ml in vitro. The data would not in‰uence im- ±0.15 mg/ml, p＜0.01). On the other hand, higher mune response in mice.16) The serum immunoglobu- doses of G. pentaphyllum signiˆcantly promoted lin levels were examined before and at the end of the more serum IgG2a (Fig. 1(C),0.44±0.03 mg/ml for experiments. The total IgG concentration in serum 0.05 G, 0.59±0.10 mg/mlfor0.50G,or0.65±0.05 did not change with or without the G. pentaphyllum mg/ml for 5.00G vs.0.35±0.01 for Normal, p＜ treatment (Fig. 1(A)). The administration of G. 0.05, p＝0.07, or p＜0.01, respectively). pentaphyllum at 0.05 or 0.5 g/kg/day enhanced the The treatment of G. pentaphyllum changed the se- serum IgG1 levels (Fig. 1(B),2.63±0.41 mg/ml for rum IgA levels similar to the eŠect of this herb to se- 0.05 G or 2.53±0.31 mg/ml for 0.50 G vs. 1.48± rum IgG1 levels (Fig. 1(D)). Nevertheless, G. pen- 0.10 mg/ml in Normal, p＝0.06 or p＜0.01, respec- taphyllum extract had dose-dependent enhancement

Fig. 1. G. pentaphyllum Extract Injected Intraperitoneally Induced Higher Serum Levels of Immunoglobulin Serum was collected from mice pre-experiment or after the injection of G. pentaphyllum for 5 consecutive days (n＝6). The mice were designated as Normal, 0.05 G, 0.50 G, and 5.00 G groups for various doses (0, 0.05, 0.50, or 5.00 g/kg/day) of G. pentaphyllum extract, respectively. The concentrations of serum IgG (A),IgG1(B), IgG2a (C),IgA(D),andIgM(E) were determined with ELISA. Data were presented as mean±standard error (SE). indicates p＜0.05, and in- dicates p＜0.01, when compared to the serum level in Normal group. 892 Vol. 127 (2007) on serum IgM concentrations (Fig. 1(E),3.77±0.43 For the production of Th2-type cytokines, all three mg/ml for 0.05 G, 4.60±0.38 mg/ml for 0.50 G, and doses enhanced IL-4 secretion from activated spleen 5.39±0.64 mg/ml for 5.00 G vs.1.08±0.12 for Nor- cells. However, the highest IL-4 production was de- mal, p＜0.05, p＜0.01, or p＜0.01, respectively). tected from 0.05G mice (Fig. 3(C),202.5±27.4 pg/ G. pentaphyllum Enhanced Immunoglobulin ml vs.65.8±11.5 pg/ml in Normal group, p＜0.05). Production from Spleen Cells The supernatants Nevertheless, the production of IL-10 showed dose- of splenocytes cultures were collected after the stimu- dependent response with the injection of G. pen- lation with 1 mg/ml of LPS for 3, 4, or 5 days. The taphyllum extract (Fig. 3(D), compared to 27.5±4.0 immunoglobulin concentrations in the supernatants pg/ml in Normal group, 48.7±4.3 pg/ml for 0.05 G, were determined with ELISA. The results in Fig. 2 p＜0.05; 71.7±8.2 pg/ml for 0.50 G, p＜0.01; and (A) indicated that higher dose of G. pentaphyllum 91.8±5.6 pg/ml for 5.00 G, p＜0.01). extract enhanced more prominent IgG secreted from DISCUSSION activated spleen cells. The culture durations slightly aŠected the production of IgG. Signiˆcant increase G. pentaphyllum is a common herbal tea in China was observed for the concentration of both IgG1 and and other Asian countries. Despite some anti-cancer IgG2a subtypes (Figs. 2(B) and 2(C)) with the and anti-infection activities, it has been described to highest dose of treatment, particularly with the IgG1 have immune modulatory eŠects for cancer patients production from 4-day culture (0.055±0.006 mg/ml and serves as a potential treatment for respiratory vs.0.011±0.001 mg/ml, p＜0.01) and IgG2a produc- in‰ammation.3,8) However, the mechanism for this tion from 5-day culture (0.081±0.005 mg/ml vs. action remains not clear. The present study demon- 0.006±0.001 mg/ml in normal, p＜0.01). In addi- strated that the extract of G. pentaphyllum enhanced tion, longer culture durations enhanced the secretion serum immunoglobulin levels and the production of of IgG1 and IgG2a. Compared to the cultures for 3 antibodies from LPS-activated spleen cells. In addi- days, the IgG1 production of 5-day culture was in- tion, the production of cytokines from Con A-stimu- creased about 2 folds from the 0.50 G and 5.00 G lated spleen cells was also improved with G. pen- mice. Similar, but more prominent pattern was ob- taphyllum treatment. served with the IgG2a production, 2.38 folds for 0.50 The signiˆcant and dose-dependent increase of se- G group and 13.18 folds from the 5.00 G group. The rum IgM levels indicates that the treatment of G. pen- dose- and culture duration-dependent production of taphyllum might improve primary immune IgA (Fig. 2(D)) and IgM (Fig. 2(E)) was also de- response.18) IgA, the most dominant immunoglobulin tected. in mucus19) and also serves as one of important medi- G. pentaphyllum Enhanced the Production of ators in the ˆrst line defense, was also enhanced with Cytokines from Activated Spleen Cells Spleen the treatment of the doses of 0.05 or 0.5 g/kg/day. cells at the concentration of 106 cells/ml were cul- This provides the preliminary evidence for the im- tured with Con A for 2 days. The supernatants were mune stimulatory activity of G. pentaphyllum.On collected and the cytokine concentrations were deter- the other hand, the extract at the doses of 0.05 or 0.5 minedwithELISA.TheresultsinFig.3(A) showed g/kg/day increased serum IgG1 and IgG2a concen- that administration of G. pentaphyllum extract en- tration. Both subtypes are considered to function as hanced the production of IFN-g from Con A-activat- adaptive or more speciˆc immune responses. It has ed spleen cells (compared to 48.3±6.3 pg/ml in Nor- been well accepted that the cytokines of Th2-type, mal group, 93.5±13.5 pg/ml for 0.05 G, p＜0.01; such as IL-4, regulate the immunoglobulin class- 112.6±8.2 pg/ml for 0.50 G, p＜0.01 or 109.7±8.7 switch and result into more IgG1 production.13) The pg/ml for 5.00 G, p＜0.01). The production of IL-2, ConA-stimulated spleen cells of G. pentaphyllum- another Th1-type cytokine, showed dose-dependent treated mice produced more IL-4 in our study. In ad- response of G. pentaphyllum administration (Fig. 3 dition, the production of IgG2a is correlated with the (B), compared to 545.9±86.5 pg/ml in Normal activity of Th1 cells.20) Therefore, the dose-dependent group, 1068.1±67.2 pg/ml for 0.05 G, p＜0.01; increase of serum IgG2a levels implicated that the ex- 1486.2±123.1 pg/ml for 0.50 G, p＜0.01; and 1751.7 tract of G. pentaphyllum might also have the activity ±59.9 g/ml for 5.00 G, p＜0.01). to enhance the Th1 activity. No. 5 893

Fig. 2. G. pentaphyllum Enhanced Immunoglobulin Production from Spleen Cells Mice were treated as described in the legend to Fig. 1. Spleen cells (5×105 cells/ml) were incubated with 1 mg/ml of LPS. The supernatants were collected after the cultures for 3, 4, and 5 days. The concentrations of IgG (A),IgG1(B),IgG2a(C),IgA(D),andIgM(E) were determined with ELISA. Date was presented as mean±SE. indicates p＜0.05, and indicates p＜0.01, when compare to the Normal group.

With the stimulation of LPS to splenocytes, more antigen,21) the results indicated that the extract of G. signiˆcant amount of IgM was produced from mice pentaphyllum should be able to activate B cells in vivo treatedwithhigherdoseofG. pentaphyllum. and pre-deposit the capacity to respond to LPS stimu- Although LPS is a typical B cell-independent lation. In addition to the similar pattern for the 894 Vol. 127 (2007)

Fig. 3. G. pentaphyllum Enhanced the Production of Cytokines from Activated Spleen Cells Mice were treated as described in the legend to Fig. 1. Spleen cells (1×106 cells/ml) were incubated with 1 mg/ml of Con A for 2 days. Culture supernatants were collected and the concentration of IFN-g (A),IL-2(B),IL-4(C),andIL-10(D) were measured by ELISA. Date was presented as mean±SE. indicates p＜ 0.05, and indicates p＜0.01, when compared to the Normal group.

production of IgA from LPS-stimulated spleen cells, hancement of IL-2 and IFN-g production from Con more dramatic production of IgGs (including total A-activated spleen cells. This might provide the IgG, IgG1, and IgG2a) from LPS-stimulated spleno- mechanism for the signiˆcant production of IgG2a cytes was observed. Since LPS itself should not have from spleen cultures. In addition to the eŠect of Th1 the ability to drive immunoglobulin gene class switch cells, G. pentaphyllum should have some eŠect on from IgM to IgG1 or IgG2a, the enhancement of Th2 cells. The production of IL-4 from Con A spleen IgG1 or IgG2a production from activated cells might cells were also increased with the treatment of G. pen- be from indirect eŠects of LPS stimulation. taphyllum at diŠerent doses. IL-4, secreted by Th2 In order to understand the eŠect of G. pentaphyl- and dendritic cells,25,26) actives B cells and promotes lum on the responses of T cells, we ˆrst measured the isotype switching to IgG1 and IgE.13) It is di‹cult to cytokine levels in serum from the treated mice. The estimate the eŠect of G. pentaphyllum extract on IgE concentrations of all tested cytokines, including IL-2, production, due to the undetectable IgE level in se- IL-4,IL-10,andIFN-g, were lower than the detecta- rum or culture supernatants in this study. The IgG1 ble levels (data no shown). Thus, we stimulated the levels in serum or activated splenocytes were in- spleen cells with Con A, a T-cell mitogen. We tested creased followed by the treatment of herbal extract, IL-2 and IFN-g as the representative cytokines for although dose-dependent responses were only ob- Th1-response. IL-2 is a T cell growth factor that pro- served from the cultures of activated splenocytes. In motes T cell proliferation.22) It can also enhance im- recently years, IL-10 was reported as the important munoglobulin synthesis and J-chain transcription in mediator of T regulatory cells that could regulate and B cell.23) IFN-g, another important cytokine secreted suppress eŠector T cells.27) T cells from mice receiving from Th1 and NK cells, is very important for anti-vi- the highest dose of G. pentaphyllum secreted most rus infection and enhances anti-tumor activity.24) The IL-10 than other groups. It is possible that high dose results in Fig. 3 indicated the dose-dependent en- of G. pentaphyllum induced some regulatory immune No. 5 895 responses. REFERENCES The chemical structures of the compounds isolated from this plant had been identiˆed to have high corre- 1) Cui J., Eneroth P., Bruhn J. S., Eur. J. lation with the ginseng .1) Recently, some Pharm. 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